Naincy R. Chandan*¹, Saji Abraham¹, Shuvasree SenGupta¹, Carole A. Parent^1,2,3,4, Alan V. Smrcka¹

¹Department of Pharmacology, University of Michigan, Ann Arbor, MI 48109, USA

²Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI

48109, USA

³Rogel Cancer Center Michigan Medicine, University of Michigan, Ann Arbor, MI 48109, USA

⁴Life Sciences Institute, University of Michigan, Ann Arbor, MI, 48109, USA.

G protein-coupled receptors (GPCRs) that couple to the Gi family of G proteins are key regulators of cell and tissue physiology. Our recent work has discovered novel roles for Gα_i in migration of neutrophils and fibrosarcoma cells downstream of activated chemoattractant receptors, but the molecular target(s) of Gα_i in these processes remain to be identified. We adopted an intact cell proximity-based labeling approach using BioID2 coupled to tandem mass tag (TMT)-based quantitative proteomics to identify proteins that selectively interact with the GTP-bound form of Gα_i1. Multiple targets were identified and validated for selective biotinylation by active BioID2-Gα_i1(Q204L), suggesting a previously unappreciated network of interactions for activated Gα_i proteins in intact cells. Extensive characterization of one candidate protein, PDZ‐RhoGEF (PRG), revealed that active-Gα_i1 strongly activates PRG. Strikingly, large differences in the ability of Gα_i1, Gα_i2, and Gα_i3 isoforms to activate PRG were observed despite over 85% sequence identity. We also demonstrate the functional relevance of the interaction between active Gα_i and PRG ex vivo in primary human neutrophils. Identification and characterization of new targets regulated by Gα_i both individually and in networks provide insights that will aid not only in investigation of diverse functional roles of Gi-coupled GPCRs in biology but also in the development of novel therapeutic approaches.

Alex S. Haimbaugh* and Tracie R. Baker

Department of Pharmacology, Wayne State University

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent and environmentally persistent endocrine disrupting chemical present in cigarette smoke and car exhaust. Our previous work demonstrated the latent reproductive effects of early-life TCDD exposure in zebrafish. Zebrafish acutely exposed to low, environmentally relevant levels of TCDD (50 pg/mL) during the two windows of sexual differentiation in development (1 hour of exposure at 3 and 7 weeks) were later infertile, showed a reduction in sperm, and exhibited gene expression consistent with an altered microenvironment, even months after exposure. Due to the highly heterogeneous cell- type and -stage landscape of the testes, we hypothesized the various cell types in the testes contribute markedly different profiles towards the pathology of TCDD exposure. To investigate the contributions of the diverse cell types in the adult zebrafish testes to TCDD-induced pathology, we turned to single-cell RNA-Seq and the 10x Genomics platform. The method successfully captured every stage of testicular germ cell development from spermatogonial stem cells to spermatozoa. We found that the testes of adult fish exposed as juveniles to TCDD contained sharply decreased populations of spermatocytes, spermatids and spermatozoa. The spermatogonia population was, in contrast, enriched following exposure. Pathway analysis supports previous findings that TCDD exposure resulted in male infertility, and suggested this outcome is due to apoptosis of spermatids and spermatozoa in response to aberrant chromatin condensation. To our knowledge, this is the first study to apply the scRNA-Seq method to the zebrafish testicular tissue.

Morteza Sarparast*¹, Elham Pourmand¹, Fan Zhang¹, Leslie Ramirez², Devon Dattmore², Benjamin Kessler², Derek Vonarx¹, Jamie K. Alan², and Kin Sing Stephen Lee^1,2

¹Department of Chemistry, Michigan State University, East Lansing, MI, USA

²Department of Pharmacology and Toxicology, Michigan State University,

East Lansing, MI, USA.

According to a United Nations report, the percentage of the population over the age of 65 is expected to increase from approximately 9% (2019), to roughly 20% by 2050. With this demographic change, we can expect a coinciding increased incidence of age-related neurodegenerative diseases (ND), including Alzheimer's Disease (AD), for which there is no cure. The two hallmark pathologies of AD are the deposition of the amyloid β (Aβ) and neurofibrillary tangles of the microtubule binding protein tau. Recent evidence suggests that increasing the epoxy-metabolites of polyunsaturated fatty acids (PUFAs) through inhibition of soluble epoxide hydrolase could be an effective strategy to mitigate AD neurodegeneration. Therefore, we sought to investigate the mechanism of the effect(s) of epoxy-PUFAs on tau- and Aβ-induced neurodegeneration using a simple biological model organism, Caenorhabditis elegans (C. elegans). The transgenic C. elegans lines expressing tau (CK1441) and Aβ (CL2355) mutants show neurodegenerative behaviors such as slow thrashing and decreased locomotion speed, respectively, and both strains exhibit hypersensitivity to serotonin, which is characteristic of these AD models. Interestingly, supplementation of an inhibitor of C. elegans sEH homologs, AUDA, rescues neurodegeneration in both the tau and Aβ transgenic strains. Our results suggest that specific epoxy-PUFAs significantly affect neurodegeneration, in particular those mediated by deposition of Aβ and/or tau. Mechanistic studies on how EH inhibition alleviates neurodegeneration induced by Aβ and/ or tau are underway.

Oluwademilade O Nuga*^1,2, Yuling Meng², Susan Irtenkauf ², Dr. Steven Brown³, Chun-Hui Lin⁴, Dr. Laila Poisson⁴, Kevin K Nelson², Dr. Ana C deCarvalho,^1,2

¹Department of Pharmacology, Wayne State University School of Medicine.

²Department of Neurosurgery, Henry Ford Hospital.

³Department of Radiation Oncology, Henry Ford Hospital.

⁴Department of Public Health, Henry Ford Hospital.

Glioblastoma (GBM) is an aggressive central nervous system tumor with a 2-year survival rate of ~15%. CDK4/6 inhibitors (CDK4/6i) are a class of targeted small molecule inhibitors approved by the FDA for treatment of distinct breast cancer subtypes. The CDK4/6-RB signaling axis is altered in over 80% of GBMs, making this class of therapeutics particularly promising in GBM treatment. However, the molecular context surrounding durable response needs to be characterized in GBMs. We employed a representative panel of GBM patient-derived cancer stem cells (CSC), to characterize their response to two CDK4/6 inhibitors (Abemaciclib and Ribociclib), complementing current measured outcomes in ongoing GBM clinical trials. Data analysis from 15 cohorts including one astrocyte control revealed 3 out of 4 lines bearing genomic amplifications in MYC or NMYC, exhibited remarkable resistance to both inhibitors. Our results show that current biomarkers such as CDK4/6 pathway hyper-activation status either by CDKN2A deletion or by CDK4/6 amplification are poor predictors. We analyzed the effect of ionizing radiation (IR) as monotherapy as well as in combination with CDK4/6i in 14 GBM CSCs and one astrocyte control. Comparison of surviving fraction revealed that combination therapy can be beneficial compared to IR treatment alone. Interestingly, in distinct subsets of CSCs, CDK4/6i were antagonistic to IR when administered concomitantly. Furthermore, CDK4/6i induced radio-resistance is mitigated if CDK4/6i is administered after IR. These findings highlight the insufficiency of current predictors of CDK4/6i response and provide much needed evidence for multimodal characterization of the molecular mechanisms driving response in GBM CSCs.

Rachel M. Golonka^1*, Beng San Yeoh¹, Piotr Czernik², Beata Lecka-Czernik², Matam Vijay-Kumar¹

¹Microbiome Consortium, Department of Physiology and Pharmacology, University of Toledo College of Medicine and Life Sciences, Toledo OH

²Micro CT and Skeletal Research Resource Core, Department of Orthopedic Surgery, University of Toledo College of Medicine and Life Sciences, Toledo OH

Hepatic Osteodystrophy is a metabolic bone disease (i.e., osteoporosis) that can later occur in patients with cholestatic liver disease. There is no suitable cholestasis animal model that allows a long-term study to examine hepatic osteodystrophy. Notably, we have generated a subset of wild-type mice to exhibit early onset anicteric cholestasis (WT^AC) as categorized by significantly elevated serum total bile acids and absence of hyperbilirubinemia at the time of weaning. In this study, we investigated how early onset cholestasis impacts liver and bone health after aging WT^AC mice to 8 months. As expected, WT^AC mice showed indicators of steatosis and mild liver cirrhosis at the gross, histological, and transcript levels. Unexpectedly, micro-computed tomography imaging and histological staining of the tibia revealed improved bone morphology in WT^AC mice. Changes in trabecular bone mass and structure included an increase of bone volume and connectivity density, switch from a rod to plate shape, and a decrease in thickness and spacing. WT^AC mice also had slightly denser cortical bone at the diaphysis but displayed less bone marrow area. Osmium tetroxide staining confirmed WT^AC mice to have lower bone marrow adiposity. Interestingly, putative genes that negatively regulate osteoclast differentiation (e.g., Rankl, Opg) were highly expressed in WT^AC mice. The significant upregulation of osteoblast (e.g., Runx2, Osx, Wnt10b) and bone mineralization (e.g., Alpl) genes in WT^AC mice further explains the potential mechanism for greater bone mass. Overall, this study delineates a novel finding that early onset cholestasis sustains bone growth even after mice reach peak bone mass.

Lyndsey Reich^1,2*, Jessica Moerland², Ana Leal², Di Zhang², Sarah Carapellucci², Carl Wagner³, Karen Liby^1,2

1. Michigan State University College of Osteopathic Medicine, East Lansing, MI

2. Pharmacology and Toxicology Department, Michigan State University, East Lansing, MI

3. School of Mathematical and Natural Sciences, Arizona State University, Glendale, AZ

Rexinoids are ligands which activate Retinoid X Receptors (RXRs), which regulate genes involved in multiple cancer-relevant processes. Rexinoids have anti-neoplastic activity with low toxicity in preclinical studies. Bexarotene, the only FDA-approved rexinoid, has been evaluated in clinical trials for metastatic breast cancer and non-small-cell lung carcinoma. Subsets of patients in these trials exhibited clinical responses despite aggressive disease refractory to prior therapy. By modifying structures of known rexinoids, we can improve potency, increase selectivity for RXR, and minimize toxicity.

We have screened a series of newly synthesized rexinoids which are selective for RXRs with minimal off-target activation. V-125 was selected as our lead based on an in vitro screening paradigm. V-125 has potent anti-inflammatory activity and a reduced tendency to increase triglycerides, a known adverse effect of rexinoids. The objective of this project was to study the anti-tumor efficacy of V-125 in the MMTV-Neu model of breast cancer.

To assess the chemopreventive effects of V-125, MMTV-Neu mice were treated with low-dose V-125 at 10 weeks of age. V-125 significantly (p < 0.001) increased the time to tumor development by ~10 weeks compared to the controls. To assess treatment efficacy, MMTV-neu mice with established tumors were treated with high-dose V-125, which significantly (p < 0.0172) increased overall survival compared to the controls. V-125 did not significantly elevate plasma triglycerides compared to control.

These studies demonstrate that our screening paradigm can predict in vivo efficacy of novel rexinoids. V-125 provides an effective therapeutic option that has potential to reduce breast cancer mortality.